Soluble cyclic analogues of beta amyloid peptide

ABSTRACT

The invention concerns a peptide having the biological activity of an inhibitor of amyloid formation and its diagnostic and medical use.

FIELD OF THE INVENTION

[0001] The present invention concerns peptides having the biologicalactivity of a modulator of amyloidogenesis, process for discoveringamyloidogenic peptides or their aggregates, pharmaceutical compositionsfor prophylaxis and therapy of diseases involving amyloidogenesis, anddiagnostic compositions to detect such diseases.

BACKGROUND OF THE INVENTION

[0002] Amyloid diseases or amyloidoses are diseases involvingamyloidogenesis, amyloid formation or aggregation of amyloid proteins incertain body tissues, often linked with cytotoxic effects. For instance,Alzheimer's disease involves amyloidogenesis of the β-amyloid peptide(β-AP).

[0003] It is assumed that the development of amyloid deposits of variouspeptides is responsible for occurrence and pathologic complications ofamyloid diseases (Lorenzo et al., Nature 368 (1994), 756-760; Lorenzo etal., Proc. Nat'l. Acad. Sci. USA 91 (1994), 12243 - 12247; Lansbury etal., Proc. Nat'l. Acad. Sci. USA 96 (1999), 3342 - 3344; Koo et al.,Proc. Nat'l. Acad. Sci. USA 96 (1999), 9989 - 9990; Sipe et al.,Critical Reviews in Clinical Laboratory Sciences 31(4) (1994), 325-354.It is further assumed that inhibitors of amyloid formation are suitablepharmaceuticals for prophylaxis and treatment of the amyloid diseases.

[0004] Linear peptides of 3 to 15 amino acids which can act as agonistsand/or inhibitors of amyloid formation are known from DE-A1 197 25 619.These peptides are distinguished by the fact that they contain at leastthe amino acids GA as the active sequence.

[0005] Peptides which modulate β-amyloid aggregation are known from U.S.Pat. No. 5,824,204. These are linearly constructed peptides of differentlengths which are derived particularly from the β-amyloid precursorprotein 770.

[0006] WO 96/39834 describes other linear peptides which can curediseases with abnormal peptide aggregation. The peptides described have3 to 15 amino acids, a hydrophobic segment of 3 amino acids and, in thishydrophobic segment, at least one amino acid which blocks a β-pleatedsheet structure.

[0007] WO 96/07425 describes neurotoxic effects of the amyloid β-proteindue to inhibiting synthetic peptides with linear structure. Diagnosisand treatment of Type II Diabetes mellitus using Amylin and derivativesof it is described n EP 0 289 287 and EP 0 309 100 A2.

[0008] Test procedures for early detection of amyloid diseases are notyet available, or are only in the development stage. That is because theproblems of protein chemistry and technical analysis caused by amyloidformation have not yet allowed any analysis of amyloid formation.Because of that, the mechanism of amyloid formation is essentiallyunexplained. As a further result, there are not at present anypharmaceutical preparations for treatment of the amyloid diseases on thebasis of modulators, especially inhibitors, of amyloidogenesis.Likewise, there are very few diagnostic procedures based on analysis ofamyloid development, such as in vitro tests for evaluation of thekinetics, quantity and quality of formation of amyloid structures. Thediagnosis still must be done symptomatically, as for Alzheimer'sdisease, for instance.

[0009] There is, in particular, need for suitable probes which candetect amyloid-forming peptides so as to determine abnormal increases inthe concentration of amyloidogenic peptides among possible diseases. Theproblem for making such probes has been that the probes themselves mustnot aggregate, but still must interact specifically, i. e., bind, withthe amyloidogenic peptides. The requirements for an inhibitor of amyloidformation will be similar. Also, such a substance obviously must notpromote aggregation or aggregate itself, and it should also be able tobind the amyloidogenic peptides specifically to prevent furtheraggregation. It has not yet been possible to utilize any of thesubstances meeting the criteria listed above in medical diagnosis ortherapy. There is, then, a great need for preparation of substanceswhich can serve both as probes for diagnosis, particularly earlydetection, of diseases involving amyloid formation and for therapy,including prophylaxis, of such diseases.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention is, therefore, based on the technicalproblem of preparing substances which can serve both as probes fordiagnosis of diseases involving amyloid formation and for therapy ofsuch diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention is explained in more detail by means of examples,sequence records, and the accompanying figures.

[0012]FIGS. 1 and 2 show electron micrographs of the fibril-formingbehavior of cβ-AP128 (1A), β-AP (1-28) (1B), [Lys¹⁷, Asp²¹] β-AP (1-28)(linear control peptide) (1C) and β-AP (1-28) (2A) and a 1:1 mixtures ofβ-AP (1-28) and cβ-AP128 (2B).

[0013]FIG. 3 shows schematically the structure of a peptide having anintramolecular bridge according to the invention and of the linearcontrol peptide.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention solves the technical problem on which it isbased by preparation of peptides with the biological activity of amodulator of amyloidogenesis, particularly an inhibitor ofamyloidogenesis, this peptide being β-AP or a derivative of it, havingat least one intramolecular bridge, and in particular at least onebridge between side chains of at least two amino acids in the peptide.In a particularly preferred embodiment of the present invention, the atleast one bridge is formed by a covalent bond between side chains of atleast two of the amino acids forming the peptide or amino acidsintroduced into the peptide (as substituents or added to the sequence).It can be provided that the bridge is formed by a direct bond betweenfunctional groups of the side chains, such as amino groups and carboxylgroups. However, it can also be provided that the side chains of theamino acids of the peptide [which are] involved in the bridging arecovalently connected to each other through a spacer or linker, that is,an aminocarboxy, diamino or dicarboxy compound.

[0015] The aggregation and the amyloid formation which causes diseasetake place through formation of an intermolecular β-pleated sheetbetween the amyloidogenic peptide sequences which is necessary fordevelopment of intermolecular hydrogen bonds and hydrophobicinteractions between the side chains of certain amino acid groups. Thisβ-pleated sheet structure results in non-covalent bonding betweeninitially two and then more amyloidogenic peptide chains, i. e., β-APmolecules, which then form insoluble aggregates, or amyloid structures.

[0016] Without the patent being bound to the theory, the intramolecularbridge in the β-AP provided according to the invention inhibits thebiological activity of a modulator of amyloidogenesis, the transitionbetween a disordered, α-helical conformation or a non-amyloidogenicstate into an aggregated or a β-pleated sheet structure which canaggregate. According to the invention, introduction of theintramolecular bridge produces a conformation restriction, i. e.,stabilization of a certain non-amyloidogenic state, such as the β-turnor the α-helix. Thus the cyclic peptides according to the invention areidentical, or very similar, in their primary structure to the wild typemolecules, the wild type β-AP, although there are major differencesbetween these peptides and the native peptides with respect to theirsecondary structures. The conformation restriction of β-AP in aconformation which cannot aggregate, attained according to theinvention, thus provides non-amyloidogenic molecules which act asaggregation inhibitors or inhibitors of amyloid development. They can,then, act as soluble agonists and probes of or for amyloid peptides. Thecyclic peptides according to the invention are distinguished by the factthat on one hand, they cannot bind specifically covalently with, orassociate specifically with, the native form, i. e., the wild type form,of the amyloid peptide on which their structure is based, and so candetect them. The peptides according to the invention are furtherdistinguished by the fact that they themselves are not amyloidogenic andat the same time, they bind to the native form of the peptide,inhibiting binding to the native form of the peptide, inhibiting itsassociation with other native peptides. In particular, binding of thecyclic peptides according to the invention occurs with soluble forms ormonomers and oligomers of the native peptide which have not yetaggregated.

[0017] The cyclic peptides according to the invention also act asagonists, probes, and inhibitors of amyloid formation, which can be usedfor diagnosis and therapy of diseases involving amyloid formation. Theycan also be used as molecular tools for analysis and investigation ofamyloidogenesis, as well as for research, development, and production ofdiagnostic and therapeutic agents for Alzheimer's disease, and forprotection against Alzheimer's disease, and also as inoculants. Theinhibition of the amyloidogenesis of the disease-causing peptides,produced according to the invention, simultaneously inhibits thecytotoxic action of the amyloid aggregate on tissue cells and eliminatesanother important pathogen. The cyclic peptides according to theinvention can be produced at high purity by chemosynthesis using currentmethods of solid-phase peptide synthesis. They have high biologicalstability, especially stability to proteolysis, which can, for example,be further increased by incorporation of unnatural amino acids and ringstructures. The cyclic peptides according to the invention can behandled easily. That is due to their high solubility, among otherthings. That contrasts with the difficulty of handling the nativemolecule, native β-AP. The cyclic peptides according to the inventionfurther exhibit little if any side effects or antigenicity when used astherapeutic agents, because the inhibitors of amyloid formation have aprimary structure which is very similar to the native peptides whichoccur in the body.

[0018] With respect to the present invention, a peptide is understood tobe a molecule having at least two amino acids linked together through anamide bond, such as an oligopeptide, a polypeptide or a protein. Thepeptide can be a naturally occurring peptide, a chemically modifiedvariant of one, or a peptide synthesized de novo. The peptide can havemodifications, such as, for instance, glycosylations or otherderivatizations such as alkylations, hydroxylations, aminations or thelike. The peptide can be altered from the naturally occurring form, i.e., the native form, and so can represent a structural and/or functionalequivalent such as a peptide analog or a peptide mimetic. Thesealterations can consist of insertions of amino acids, exchanges of aminoacids, amino acid inversions, amino acid additions and/or amino aciddeletions. The peptide can also contain unusual and/or unnatural aminoacids. Likewise, the amino acids of the peptide can occur in the D- or Lconfiguration.

[0019] In connection with the current invention, a peptide having thebiological activity of a modulator of amyloidogenesis is understood tobe a peptide which can influence peptides which form amyloid oramyloid-like structures with respect to their ability to aggregate intoamyloids, particularly to promote, initiate or inhibit, for instance, toreduce or block amyloidogenesis. Promotion of the ability to aggregatecan be desirable for research or for the deliberate tissue-specificlocalization of amyloids in tissues in which they are not harmful. Apeptide having the biological activity of a modulator of amyloidogenesisis particularly distinguished by the fact that this peptide can interactwith amyloidogenic peptides in such a way that their ability toaggregate is altered, and in particular, inhibited, especially withrespect to the rate of aggregation, i.e., the velocity of aggregation,and the size or amount of the aggregates formed. This alteration of theaggregation ability occurs preferably by association or attachment,preferably through non-covalent bonding, to the preferably soluble formof the amyloidogenic peptide or to a soluble oligomer of theamyloidogenic peptide. In a particularly preferred embodiment of thepresent invention an inhibitory effect is an effect by which the lagphase of aggregation is extended, in comparison with the lag phase ofonly naturally occurring amyloidogenic peptides, by a factor of at least1.5; 1.8; 2; 2.5; 3; 4; 5; 8; 10; 20; 40; 50; or 100. In a furtherembodiment of the invention there is an inhibitory effect if the degreeof aggregation, compared with that of only naturally occurringamyloidogenic peptides or aggregates formed from them is reduced by 10,20, 30, 40, 50, 60, 70, 80, 90 or 100%.

[0020] In connection with the present invention, the concept β-AP isunderstood to be a peptide having the biological activity of a modulatorof amyloidogenesis, having an intramolecular bridge and primarystructure identical to the wild type sequence, or a different primarystructure, i. e., amino acid sequence. Commonly known test procedures,such as are described in U.S. Pat. No. 5,854,204, can be used todetermine the modulation of amyloidogenesis, of the rate of aggregation,of the degree of aggregation (total amount of aggregation) and thevelocity of or capability for aggregation. That patent, combined withthe test procedures described there, are included in the disclosure ofthe present teaching.

[0021] In connection with the present invention, the concept “aminoacids participating in bridge formation” is understood to mean thoseamino acids of the peptide, the side chains of which have the functionalgroups of which are directly or indirectly linked together to make abond leading to the intramolecular bridge. Therefore the amino acidswhich participate in bridge formation have side chains with functionalgroups which either link directly with other functional groups of adifferent side chain of a different amino acid of the peptide, or theirfunctional groups are linked with at least one spacer molecule which inturn is linked with the functional group of a side chain of anotheramino acid of the peptide which participates in the bridge formation.Such functional groups can, for instance, be hydroxy, amino, carboxyand/or thiol groups. The amino acids participating in the bridgeformation can, naturally, be amino acids existing in the native sequenceat this position. It is also possible according to the invention tointroduce substituting amino acids which do not naturally occur at thissite, or unnatural or unusual amino acids, by suitable substitutions ofthe naturally occurring amino acids. Those then are used for or involvedin the bridge formation. Obviously it is also possible according to theinvention for the amino acids participating in bridge formation also tobe introduced into the amino acid sequence of the β-AP without anaturally occurring amino acid being deleted. Introduction of aminoacids which do not occur naturally at this position can have theadvantage that specific functional groups which allow or simplify bridgeformation can be introduced deliberately at this position. One preferredembodiment of the invention provides for introduction into the aminoacid sequence of amino acids which participate in bridge formation: Dab(2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Ser, Asp,Glu, Cys, Lys and/or Orn, such as by substitution or addition.

[0022] One advantageous development of the invention provides that theamino acids of the peptide which participate in bridge formation,preferably the two amino acids of the peptide which participate inbridge formation are arranged at relative separations of i+3, i+4, i+5,i+6 or i+7 in the peptide.

[0023] In one preferred embodiment of the present invention it isprovided that the side chains of the amino acids of the peptide whichparticipate in bridge formation are linked together by a spacer, withthe spacer selected from the group consisting of X—(CH₂)_(n)—Y with n=1to 6 and X/Y: NH₂/COOH or COOH/NH₂ or NH₂/NH₂ or COOH/COOH or OH/OH orSH/SH; or X is one of NH₂ or COOH or OH or SH, and Y is one of COOH orNH₂ or SH or OH. Obviously it is also possible to use other spacers. Itis important according to the invention that that nature of X and Y,that is, of the terminal functional groups of the spacer or linker, bematched to the functional groups of the side chains being linked so thatthey can enter into a covalent chemical bond, such as by linking anamino group with a carboxyl group, or a carboxyl group with a hydroxylgroup or two hydroxyl groups or two SH groups.

[0024] In another preferred embodiment the cyclic peptide of the presentinvention can have other functional groups which are not related tobridge formation but which serve, for instance, for immobilization onsupports, interaction with other molecules, detection, or the like. Suchgroups can be functionalized alkyl groups, aromatic groups, glycogroups, lipid groups, cyclic, heterocyclic or polycyclic groups,biotin-containing groups, avidin-containing groups,streptavidin-containing groups or the like. Obviously it can also beprovided that peptides of the present invention fuse with otherpeptides, polypeptides or proteins or fragments of them, for example,with wild type β-AP, or derivatives or fragments of it.

[0025] The peptides according to the invention can have marker groups,such as enzymes, prosthetic groups, fluorescent materials, radioactivematerials or luminescent materials.

[0026] Horseradish peroxidase, alkaline phosphatase, β-galactosidase oracetylcholinesterase are examples of enzymes which can be used.Streptavidin/biotin and avidin/biotin are examples of prosthetic groups.Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin areexamples of fluorescent material which can be used. Examples ofradioactive materials which can be used include ¹⁴C, 123I, ¹²⁴I, ¹²⁵I,¹³¹I, ^(99m)Tc, ³⁵S or ³H.

[0027] The amino acid numberings and positions used in the presentteaching refer to the sequence of the native wild type β-AP, as isdescribed in Table 1 of Hilblich et al., (J. Mol. Biol. 228 (1992),460-473. The contents disclosed in that publication are completelyincluded in the disclosure of the present teaching with respect to thesequence of the amino acids of β-AP and its preparation, and it isclearly stated that protection for that is requested in the context ofthe invention. The numbering, according to current teaching, is alwaysfrom the N terminus to the C terminus.

[0028] In another particularly preferred embodiment of the presentinvention, in which the original peptide, i. e., the stem peptide, isβ-AP, the amino acids participating in bridge formation appear in theregions 15 to 24 of the β-AP. According to another preferred developmentof this embodiment which relates back to the β-AP stem peptide, the β-APhas 1 to 28 (β-AP 1-28 or β-AP128), 1 to 40 (β-AP 1 to 40 or β-AP140), 1to 42 (β-AP 1-42 or βAP142) or 1 to 43 (β-AP 1-43 or β-AP143) aminoacids. In another preferred embodiment of the present invention theamino acids which participate in the bridge formation of the β-AP arethose which are naturally there or are there for example as amino acidsintroduced by substitution, e. g., lysine and aspartic acids, forexample, at positions 17 and 21.

[0029] In one particularly preferred embodiment of the present inventionthe invention concerns the cyclic peptide cyclo^(17,21) [Lys¹⁷, Asp²¹]β-AP (1 to 28) (abbreviated as cβ-AP 128 or cβ-AP (1 to 28). The aminoacid sequence is shown in SEQ ID No. 2.

[0030] In another preferred embodiment, the cyclic peptide iscyclo^(17,21) [Lys¹⁷, Asp²¹] β-AP (1 to 40) (abbreviated as: cβ-AP 140).The amino acid sequence is shown in SEQ ID No. 3.

[0031] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Lys¹⁷, Asp21] β-AP (1 to 42) (abbreviated as cβ-AP 142).The amino acid sequence is shown in SEQ ID No. 4.

[0032] In one preferred embodiment the cyclic peptide is cyclo^(17,21)[Asp¹⁷ Lys²¹] β-AP (1 to 28). The amino acid sequence is shown in SEQ IDNo. 5.

[0033] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Asp¹⁷, Lys²¹] β-AP (1 to 40). The amino acid sequence isshown in SEQ ID No. 6.

[0034] In another preferred embodiment the cyclic peptide iscyclo^(17,21)[Asp¹⁷, Lys²¹] β-AP (1 to 42). The amino acid sequence isshown in SEQ ID No.7.

[0035] In another particularly preferred embodiment the cyclic peptideis cyclo^(17,21) [Asp¹⁷, Orn²¹] β-AP (1 to 28) (Orn: ornithine). Theamino acid sequence is shown in SEQ ID No.8.

[0036] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Asp¹⁷, Orn²¹] β-AP (1 to 40). The amino acid sequence isshown in SEQ ID No.9.

[0037] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Asp¹⁷, Orn²¹] β-AP (1 to 42). The amino acid sequence isshown in SEQ ID No.10.

[0038] In a particularly preferred embodiment of the present inventionthe cyclic peptide is cyclo^(17,21) [Asp¹⁷, Dab²¹] β-AP (1 to 28) (Dab:2,4-diaminobutyric acid). The amino acid sequence is shown in SEQ IDNo.11.

[0039] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Asp¹⁷, Dab²¹] β-AP (1 to 40). The amino acid sequence isshown in SEQ ID No.12.

[0040] In another preferred embodiment the cyclic peptide iscyclo^(17,21) [Asp¹⁷, Dab²¹] β-AP (1 to 42). The amino acid sequence isshown in SEQ ID No. 13.

[0041] In a particularly preferred embodiment of the present inventionthe cyclic peptide is cyclo^(17,21) [Orn¹⁷, Asp21] β-AP (1 to 28). Theamino acid sequence is shown in SEQ ID No.14.

[0042] In another preferred embodiment the cyclic peptide iscyclo^(17,21)[Orn¹⁷, Asp21] β-AP (1 to 40). The amino acid sequence isshown in SEQ ID No.15.

[0043] In another preferred embodiment the cyclic peptide iscyclo^(17,21)[Orn¹⁷, Asp²¹] β-AP (1 to 42). The amino acid sequence isshown in SEQ ID No.16.

[0044] In another particularly preferred embodiment the cyclic peptideis cyclo^(17,21)[Dab¹⁷, Asp²¹] β-AP (1 to 28). The amino acid sequenceis shown in SEQ ID No.17.

[0045] In another preferred embodiment the cyclic peptide iscyclo^(17,21)[Dab¹⁷, Asp²¹] β-AP (1 to 40). The amino acid sequence isshown in SEQ ID No. 18.

[0046] In another preferred embodiment the cyclic peptide iscyclo^(17,21)[Dab¹⁷, Asp²¹] β-AP (1 to 42. The amino acid sequence isshown in SEQ ID No.19.

[0047] The invention also concerns antibodies, especially monoclonal orpolyclonal antibodies which can specifically recognize the cyclicpeptide of the invention and bind to it. The antibodies can be modifiedin the usual manner, e. g., labeled. They can also occur in theimmobilized form, on a carrier or fixed to beads.

[0048] The invention also provides for processes by which the peptidesaccording to the invention are used as antigens to immunize human oranimal organisms and the antibodies formed are obtained. Thus theinvention also concerns processes for obtaining monoclonal andpolyclonal antibodies in which the peptides according to the inventionare used as antigens, particularly introduced into human or animalorganisms and the antibodies formed after immunization are obtained orcells producing antibodies, such as spleen cells, are obtained toproduce hybridoma cells producing monoclonal antibodies. Therefore theinvention also concerns use of the peptides according to the inventionto obtain monoclonal or polyclonal antibodies by means of processeswhich are themselves known. This can also involve recombinant processes,thus, for example, production of antibodies in E. coli. The antibodiesobtained, which can, therefore, also be human or humanized antibodies,can be used for research, e. g., as a research tool, or for therapeuticor diagnostic purposes, especially for diagnosis and therapy ofAlzheimer's disease. The polyclonal or monoclonal antibodies accordingto the invention can serve, for example, for analysis of the course ofthe disease in patients treated with peptides according to theinvention, for instance, or for isolation and identification of othertherapeutically effective peptides. The peptides according to theinvention prove to be particularly advantageous with respect to theiruse as immunogens for production of antibodies for diagnostic andtherapeutic purposes because of their substantially improved ease ofhandling compared with the naturally occurring poorly soluble analogs.The antibodies produced in this manner can in one embodiment detectspecifically the peptides according to the invention and, in anotherembodiment, can also detect the native wild type β-AP present, possiblyin the aggregated form, so that the antibodies according to theinvention can, for instance, be used to diagnose Alzheimer's disease.The invention also concerns processes for immunizing human or animalorganisms, by which the peptide according to the invention is applied tohuman or animal organisms and immunization is achieved against β-AP orits derivatives.

[0049] The peptides according to the invention can be synthesized and/ormodified chemically in the usual manner. They can also be produced byrecombinant DNA technology, or isolated from natural sources andmodified.

[0050] The invention also concerns pharmaceutical compositionscontaining a cyclic peptide designated above in pharmaceutically activeamount and in a pharmaceutically acceptable carrier. In one embodimentof the present invention, the pharmaceutical composition contains atleast one cyclic peptide of the present invention in a prophylacticallyor therapeutically effective amount which is sufficient to alter, and inparticular, to inhibit, aggregation, especially the rate of aggregationand/or the quantity of aggregate formed, [or] to alter native amyloidpeptides, in particular, to inhibit them. In another embodiment of thepresent invention, the pharmaceutical composition of the presentinvention has at least one cyclic peptide of the present invention in aprophylactically or therapeutically effective amount which is sufficientto alter, and in particular, to inhibit the neurotoxic effect ofnaturally occurring amyloidal peptides or their aggregates. Thepharmaceutically active amount depends on various factors, such as thestate of the disease, the size of the patient, the weight of thepatient, the amount of endogenous amyloidal peptides present, etc.

[0051] Examples of pharmaceutically active carriers include solvents,dispersing agents, coatings, fillers, antibacterial and antifungalagents, isotonic agents, etc. Other additives can optionally be added,such as colorings or flavorings, emulsifiers, binders, release agents,etc.

[0052] Another embodiment of the present invention can provide foradministering the pharmaceutical composition intravenously, orally,intraperitoneally, intraspinally, intracerebrally or intramuscularly.Provision can be made for administering the composition in the form of asterile aqueous solution or dispersion, or a powder.

[0053] Provision can optionally be made for using other medicallyeffective substances along with the cyclic peptide of the presentinvention in the pharmaceutical composition. Provision can be made forutilizing other substances in the pharmaceutical composition whichserve, for instance, for transport in the target organism, e. g.,through the blood-brain barrier.

[0054] In another preferred embodiment, the present invention concernsthese diagnostic compositions containing at least one cyclic peptide ofthe present invention which preferably has a detection marker. Such adetection marker can be a radioactive label such as radioactive iodineor technetium. However, the detection marker can also be a fluorescentlabel, luminescent or enzyme label, or a spin label.

[0055] In another preferred embodiment, the invention concerns a processfor detecting, especially screening, scanning or detecting,amyloidogenic peptides, especially β-AP or its derivatives, oligomers ofamyloidogenic peptides or aggregates of amyloidogenic peptides, and ofantibodies against such materials in a biological sample or an animal orhuman organism. In such a process at least one cyclic peptide of thepresent invention, preferably having a detection marker, or an antibodyof the present invention with a detection marker as a probe is broughtinto contact with a sample being examined, preferably in a liquid, andan association of the peptide with amyloidogenic peptides, oligomers ofthem, aggregates of them, or antibodies against them is detectedqualitatively and/or quantitatively. This process can be carried out invitro in one embodiment of the present invention, and in vivo in anotherembodiment. Thus the concept of ‘bringing into contact’ includesincubation of the cyclic peptide with a sample in vitro or introductionon the cyclic peptide to a site in vivo where, for instance, naturalamyloid peptide or its aggregate is present. Obviously, provision isalso made to carry out this process in the presence of at least oneother test substance such as native β-AP, other peptide variants,potential pharmaceuticals, potential diagnostic agents or markersubstances in the form, for example, of competitive test procedures sothat the effect of these other test substances on the aggregationbehavior of natural β-AP and/or peptides according to the invention canbe detected.

[0056] Provision can be made according to the invention for using thecyclic peptides in immobilized form. So provision can be made in apreferred manner to give the cyclic peptide other functional groupswhich make possible immobilization on solid carriers. The immobilizationcan, for instance, be done using the peptides according to the inventionin antibody-based test, screening and analytical procedures, e. g., foridentifying antibodies specific for β-AP or β-AP derivative. Theimmobilization can also be used for preparative processes in researchand therapy such as for affinity matrices for binding, isolation, anddepletion of amyloids from various liquids.

[0057] The invention also concerns the use of at least one cyclicpeptide of the present invention for producing a medication of diseasescharacterized by amyloid development, i. e., amyloidoses, especiallyprimary, secondary, inherited and/or isolated amyloidoses such asAlzheimer's disease, especially sporadic or familial Alzheimer'sdisease.

[0058] Therefore the invention also concerns processes for diagnosisand/or therapy, for instance, prophylaxis of amyloidoses, by which atleast one cyclic peptide of the present invention is applied to a humanor animal body or an isolated component of such a body in apharmaceutically or diagnostically effective amount and, in the case ofdiagnostic use, bonding to amyloid structures is detected.

[0059] The invention also concerns processes for altering theaggregation of amyloidogenic peptides, oligomers or aggregates of them,or for inhibiting the cytotoxicity of amyloidogenic peptides, oligomersor aggregates of them, whereby peptides of the present invention arebrought into contact, in vivo or in vitro with the amyloidogenicpeptides, oligomers or aggregates of them and the aggregation behaviorof the amyloidogenic peptides, oligomers or aggregates of them isaltered, and especially the aggregation is reduced or inhibited. In thesense of the present invention, an amyloidosis is especially Alzheimer'sdisease.

[0060] The invention particularly concerns a process for modifying,preferably preventing, formation of aggregate from an amyloid β-APcontained in a liquid, whereby a peptide of the present invention isbrought into contact with the liquid and incubated. This process canalso serve for depleting or removing amyloid from liquid, e. g., bodyfluids.

[0061] The present invention also concerns kits comprising the cyclicpeptides according to the invention and/or monoclonal or polyclonalantibodies directed against them, in which these agents can optionallybe labeled.

[0062] Other advantageous embodiments of the invention arise from thesub-claims.

[0063] The sequence record which is part of the present teaching shows:

[0064] SEQ ID No. 1: the wild type amino acid sequence of β-AP (1-43),

[0065] SEQ ID Nos. 2 - 19: the amino acid sequences according to theinvention, where there is in each case an intramolecular bridge betweenthe side chains of the amino acids 17 and 21.

EXAMPLE 1

[0066] Preparation, Purification and Characterization of cβ-AP128 andControl β-AP (1-28)

[0067] The peptides were produced by current methods of solid-phasepeptide synthesis using the Boc/Bzl synthesis strategy on PAM resin(Barany, G., Kneib-Cordonier, N. and Mullen, D. G. (1987), J. Pept. Res.30, 705-739. They were removed from the resin with HF/anisole (10:1),and purified by preparative HPLC on a C18 column (Kapurniotu & Taylor(1995) J. Med. Chem. 38, 836-847). For the synthesis of cβ-AP128, theside chains of Lys¹⁷ and Asp²¹ were protected by Fmoc orFluorenyl-methyl ester (Ofm) protective groups, which were then removedselectively with 20% piperidine/DMF before forming the lactam bridge(Kapurniotu & Taylor, J. Med. Chem. 38, 836-847 (1995)). The sidechain-to-side chain cyclization was done withbenzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate(BOP). It was tested for completeness with the Kaiser test (Kapurniotu &Taylor, J. Med. Chem. 38, 836-847 (1995)). In order to get a completecyclization reaction, the cyclization reaction was done four times inmixtures of DMF, NMP and DMSO. (The duration of a cyclization was about20 hours). The masses of the products purified by HPLC were determinedby MALDI-MS, and their purity was verified by analytical HPLC.

EXAMPLE 2

[0068] cβ-AP128: Aggregation; testing of the fibril formation; testingof the inhibitory action on aggregation of β-AP (1-28).

[0069] Physicochemical Properties of cβ-AP128 and Testing the FibrilFormation

[0070] The physicochemical properties of cβ-AP were investigated byFT-IR (Fourier-transform infrared spectrometry) and CD (circulardichroism spectropolarimetry). EM (electron microscopy) and AFM (atomicforce microscopy) were used to test the properties of fibrils ofcβ-AP128. In the EM studies, 450 μM solutions of cβ-AP128 or β-AP (1-28)or [Lys¹⁷, Asp²¹] β-AP(1-28) in 10 mM phosphate buffer, 150 mM NaCl and30% acetonitrile, pH 5.5 were initially incubated for several days andtested for fibril formation. These studies showed that highlyconcentrated solutions of cβ-AP128 did not form any fibrils over manydays (more than 30) (FIG. 1A). That is in contrast with the controlpeptides β-AP (1-28) (naturally occurring sequence) and [Lys¹⁷, Asp²¹]β-AP (1-28) (linear control peptide), which are completely fibrillatedafter three days incubation (FIGS. 1B, 1C).

[0071] FT-IR of the solutions/suspensions above showed that the bridgingintroduced in cβ-AP128 prevents the β-pleated sheet conformation whichnative β-AP and the control peptide [Lys¹⁷, Asp²¹] β-AP (1-28) take onafter aging, and thus inhibits aggregation and fibril formation.

[0072]FIG. 3A shows the native amino acid sequence of the β-AP fragmentβ-AP (1-28). Positions 17 to 20 are emphasized to show their importantrole in the association and fibril formation. The upper part of FIG. 3Bshows schematically the structure of the inventive β-AP (1-28)derivative cyclo^(17,21)[Lys¹⁷, Asp²¹] β-AP (1-28), which is abbreviated‘cyclo-β-AP(1-28)’ or called cβ-AP128, with the intramolecular bridgebetween the side chains of Lys¹⁷ and Asp²¹, particularly the ε-aminogroup of Lys and the β-carboxyl group of Asp. Therefore this example hasan intramolecular bridge, in which the side chains of the amino acids atpositions 17 and 21 are covalendy linked through a lactam bond, withoutintermediate linkers or spacers. The lower part of FIG. 3B shows thecontrol peptide, control-β-AP(1-28), i. e., [Lys¹⁷, Asp21] β-AP (1-28).The figure makes it clear that replacement of the leucine at position 17by lysine and of alanine at position 21 by aspartic acid yields analogswhich differ from the native β-AP (1-28) peptide and the covalentbonding of the side chains of these substituents, through the lactambridge introduced between the ε-amino group and the β-carboxyl group,gives the structure according to the invention.

EXAMPLE 3

[0073] Inhibition of the Aggregation and Fibril Formation of β-AP (1-28)or [Lys¹⁷, Asp²¹] β-AP (1-28) by cβ-AP128

[0074] The effect of cβ-AP128 on the aggregation and fibril formation ofβ-AP (1-28) was tested by CD and EM. A solution of 450 μM β-AP (1-28)and cβ-AP128 in 10 mM phosphate buffer, 150 mM NaCl and 30%acetonitrile, pH 5.5, was incubated for several days and tested forfibril formation by EM (FIGS. 2A and 2B). CD showed no changes of the CDspectra of these mixtures for more than 1 month, although β-AP alone or[Lys¹⁷, Asp²¹] β-AP (1-28) alone formed β-pleated sheets in less than 10hours and then precipitated out as insoluble amyloid.

1 19 1 43 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr GluVal His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile AlaThr 35 40 2 28 PRT Homo sapiens PEPTIDE (17)..(21) The side chains ofthe amino acids at positions 17 and 21 are linked together through anintramolecular bridge. 2 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu ValHis His Gln Lys 1 5 10 15 Lys Val Phe Phe Asp Glu Asp Val Gly Ser AsnLys 20 25 3 40 PRT Homo sapiens PEPTIDE (17)..(21) The side chains ofthe amino acids at positions 17 and 21 are linked together through anintramolecular bridge. 3 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu ValHis His Gln Lys 1 5 10 15 Lys Val Phe Phe Asp Glu Asp Val Gly Ser AsnLys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40 4 42PRT Homo sapiens PEPTIDE (17)..(21) The side chains of the amino acidsat positions 17 and 21 are linked together through an intramolecularbridge. 4 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys 1 5 10 15 Lys Val Phe Phe Asp Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 5 28 PRTHomo sapiens PEPTIDE (17)..(21) The side chains of the amino acids atpositions 17 and 21 are linked together through an intramolecularbridge. 5 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys 1 5 10 15 Asp Val Phe Phe Lys Glu Asp Val Gly Ser Asn Lys 20 25 6 40PRT Homo sapiens PEPTIDE (17)..(21) The side chains of the amino acidsat positions 17 and 21 are linked together through an intramolecularbridge. 6 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys 1 5 10 15 Asp Val Phe Phe Lys Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40 7 42 PRT Homosapiens PEPTIDE (17)..(21) The side chains of the amino acids atpositions 17 and 21 are linked together through an intramolecularbridge. 7 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys 1 5 10 15 Asp Val Phe Phe Lys Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 8 28 PRTHomo sapiens PEPTIDE (21) Xaa=Orn 8 Asp Ala Glu Phe Arg His Asp Ser GlyTyr Glu Val His His Gln Lys 1 5 10 15 Asp Val Phe Phe Xaa Glu Asp ValGly Ser Asn Lys 20 25 9 40 PRT Homo sapiens PEPTIDE (21) Xaa=Orn 9 AspAla Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15Asp Val Phe Phe Xaa Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 40 10 42 PRT Homo sapiens PEPTIDE(21) Xaa=Orn 10 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln Lys 1 5 10 15 Asp Val Phe Phe Xaa Glu Asp Val Gly Ser Asn Lys GlyAla Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 11 28PRT Homo sapiens PEPTIDE (21) Xaa=Dab 11 Asp Ala Glu Phe Arg His Asp SerGly Tyr Glu Val His His Gln Lys 1 5 10 15 Asp Val Phe Phe Xaa Glu AspVal Gly Ser Asn Lys 20 25 12 40 PRT Homo sapiens PEPTIDE (21) Xaa=Dab 12Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 1015 Asp Val Phe Phe Xaa Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 2530 Gly Leu Met Val Gly Gly Val Val 35 40 13 42 PRT Homo sapiens PEPTIDE(21) Xaa=Dab 13 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln Lys 1 5 10 15 Asp Val Phe Phe Xaa Glu Asp Val Gly Ser Asn Lys GlyAla Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 14 28PRT Homo sapiens PEPTIDE (17) Xaa=Orn 14 Asp Ala Glu Phe Arg His Asp SerGly Tyr Glu Val His His Gln Lys 1 5 10 15 Xaa Val Phe Phe Asp Glu AspVal Gly Ser Asn Lys 20 25 15 40 PRT Homo sapiens PEPTIDE (17) Xaa=Orn 15Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 1015 Xaa Val Phe Phe Asp Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 2530 Gly Leu Met Val Gly Gly Val Val 35 40 16 42 PRT Homo sapiens PEPTIDE(17) Xaa=Orn 16 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln Lys 1 5 10 15 Xaa Val Phe Phe Asp Glu Asp Val Gly Ser Asn Lys GlyAla Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 17 28PRT Homo sapiens PEPTIDE (17) Xaa=Dab 17 Asp Ala Glu Phe Arg His Asp SerGly Tyr Glu Val His His Gln Lys 1 5 10 15 Xaa Val Phe Phe Asp Glu AspVal Gly Ser Asn Lys 20 25 18 40 PRT Homo sapiens PEPTIDE (17) Xaa=Dab 18Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 1015 Xaa Val Phe Phe Asp Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 2530 Gly Leu Met Val Gly Gly Val Val 35 40 19 42 PRT Homo sapiens PEPTIDE(17) Xaa=Dab 19 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln Lys 1 5 10 15 Xaa Val Phe Phe Asp Glu Asp Val Gly Ser Asn Lys GlyAla Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40

1. Peptide with the biological activity of a modulator ofamyloidogenesis, characterized in that the peptide is β-AP (β-amyloidpeptide) and has at least one intramolecular bridge.
 2. Peptideaccording to claim 1, characterized in that the modulator is aninhibitor.
 3. Peptide according to one of the foregoing claims, in whichthe bridge is formed by covalent bonding between side chains of theamino acids forming the peptide.
 4. Peptide according to one of theforegoing claims, in which the amino acids of the peptide whichparticipate in bridge formation are arranged at relative distances ofi+3, i+4, i+5, i+6 or i+7.
 5. Peptide according to one of the foregoingclaims, in which the side chains of the amino acids which participate inbridge formation are linked together covalently directly or through aspacer.
 6. Peptide according to one of the foregoing claims, in whichthe spacer is an aminocarboxy, diamino, or dicarboxy compound. 7.Peptide according to one of the foregoing claims; in which the aminoacids which participate in bridge formation are the amino acids whichoccur naturally at these positions of the peptide.
 8. Peptide accordingto one of claims 1 to 6, in which at least one of the amino acids whichparticipate in bridge formation does not occur naturally at thisposition.
 9. Peptide according to claim 8, in which the at least oneamino acid which participates in the bridge formation is a substituentof the amino acid which occurs there naturally or is an added introducedamino acid.
 10. Peptide according to claim 8 or 9, in which the aminoacids which participate in bridge formation are arranged in the vicinityof positions 15 to 24 of the wild type β-AP.
 11. Peptide according toclaim 10, in which β-AP consists of the amino acids 1 to 28, 1 to 40 or1 to 42 or 1 to
 43. 12. Peptide according to one of claims 10 or 11, inwhich the amino acids which participate in bridge formation are theamino acids at positions 17 and
 21. 13. Peptide according to one ofclaims 10 to 12, in which the peptide having the biological activity ofa modulator is a peptide selected from the group consisting of thepeptides with the amino acid sequences shown in SEQ ID Nos. 2 to
 19. 14.Peptide according to one of the foregoing claims, in which the peptideis derivatized by at least one functional group, selected from the groupconsisting of an alkyl group, functionalized alkyl group, aromaticgroup, oligopeptide, polypeptide, glyco group and lipid group. 15.Peptide according to one of the foregoing claims, in which the peptideis immobilized on a carrier.
 16. Antibody which specifically detects apeptide according to one of claims 1 to
 15. 17. Antibody according toclaim 16 which is a monoclonal or polyclonal antibody. 18.Pharmaceutical composition containing a peptide according to one ofclaims 1 to 15 and a pharmaceutically acceptable carrier.
 19. Diagnosticcomposition containing a peptide having a detection marker according toone of claims 1 to 15 or an antibody according to claim 16 or
 17. 20.Diagnostic composition according to claim 19, in which the detectionmarker is a radioactive label, a fluorescent label, an enzyme label, aluminescence label or a spin label.
 21. Process for qualitative and/orquantitative detection of amyloidogenic β-AP or aggregates of it, inwhich a peptide having a detection marker according to one of claims 1to 15, or an antibody having a detection marker according to one ofclaims 16 or 17, as a probe, is brought into contact with a sample to beexamined and binding of the peptide to amyloidogenic β-AP peptides ortheir aggregates which are possibly present is detected.
 22. Process formodification, especially prevention, of aggregate formation fromamyloidogenic β-AP in a liquid, in which a peptide according to one ofclaims 1 to 15 is brought into contact with the liquid and incubated.23. Use of a peptide according to one of claims 1 to 15 and/or anantibody according to one of claims 16 or 17 to produce a medication forprophylaxis and/or treatment of diseases characterized by amyloidformation, especially Alzheimer's disease.
 24. Use of a peptideaccording to one of claims 1 to 15 as an immunogen with better handlingcharacteristics for production of antibodies for diagnostic, therapeuticand research purposes.
 25. Process according to claim 22 for preparativedepletion of amyloid from liquids, especially body fluids.